PROJECT SUMMARY Background: Many steps of cancer drug development involves the use of standard cancer cell lines (SCCL) which have been instrumental in the development of many targeted therapies; including Herceptin, Gleevac and Tamoxifen among others. However, establishing new CCL that mimic human disease is still challenging due to in vitro clonal selection and loss of the original tumor phenotype. In addition, the efficiency of creating new cell lines is very low. As a result, ~90% of ovarian and breast cancer research has been carried out in 5 CCL each 1, 2. These SCCL and leukemia cell lines do not fully represent the real-life diversity of human disease3-5. Innovation: One of the persistent shortcomings of cell culture technology is the use of full serum, drugs and feeder layers that are partly responsible for the poor replication of the original tumor phenotype. In order to address these problems we developed a series of serum-free tissue specific culture (TSC) media that increases the efficiency of establishing new cell lines to > 50% and retains important tissue specific original tumor features. Preliminary data: We recently published the first application of the TSC technology for ovarian cancer (OvCa), describing 25 new OvCa cell lines that retain molecular, histologic and outcome features of the patient tumors6. Objectives: Our long-term goal is to develop novel TSC media and methods to expand our technology to all tumor types and improve the methods to solve the stromal and normal cell overgrowth problem. Specific Aims: While we work on all tumor types, in this grant we focus on solid tumors (Breast, Ovary) and a liquid tumor (Leukemia) to illustrate that our system can be adapted to culture the full spectrum of tumor types. Aim 1) Characterization of new breast cancer cell lines: We formulated a new normal breast specific and breast cancer specific media, which we will use to establish matching normal and cancer cell lines. Aim 2) Characterization of new acute myeloid leukemia (AML) lines: We formulated a new serum-free medium customized for AML, which we will use to establish new AML cell lines. Aim 3) Culture of sub-optimal samples: Tissue samples with <10% tumor are difficult to culture due to stromal over-growth. We developed a method that suppress stroma to enable culture of sub-optimal tissue samples. Validation of Cancer-Relevant Biospecimen Science Technology: The genome, transcriptome and proteome of each cell line will be compared to the original patient tumor, existing cell lines and tumor datasets. Substantial Improvement and New Capabilities. Our TSC system can maintain cell lines without feeder layers, drugs, extracts, and suppresses the over-growth of stromal and normal cells. Transformative Potential. The biological relevance of SCCL is highly questionable. Hence, it is not surprising that drugs that are developed using SCCL frequently fail in the clinic. Our ability to provide biologically relevant BrCa, OvCa, and AML cell lines can have a transformative effect on cancer drug development.